biomolecules

Article
Differential Plasma Expression Profiles of Long
Non-Coding RNAs Reveal Potential Biomarkers for
Systemic Lupus Erythematosus
Guo-Cui Wu 1,† , Yan Hu 2,3,† , Shi-Yang Guan 2,3 , Dong-Qing Ye 2,3, * and
Hai-Feng Pan 2,3, *
1 School of Nursing, Anhui Medical University, 15 Feicui Road, Hefei 230601, China; gcwu82@126.com
2 Department of Epidemiology and Biostatistics, School of Public Health, Anhui Medical University,
81 Meishan Road, Hefei 230032, China; huyan1104@126.com (Y.H.); gsy92@foxmail.com (S.-Y.G.)
3 Anhui Province Key Laboratory of Major Autoimmune Diseases, 81 Meishan Road, Hefei 230032, China
* Correspondence: ydq@ahmu.edu.cn (D-Q.Y.); panhaifeng@ahmu.edu.cn (H-F.P.);
Tel.: +86-551-65161165 (D-Q.Y.); Fax: +86-551-65161171 (D-Q.Y.)
† These authors contributed equally to this work.




Received: 30 March 2019; Accepted: 20 May 2019; Published: 28 May 2019


Abstract: Identify long non-coding RNAs (lncRNAs) that might serve as biomarkers for systemic lupus
erythematosus (SLE) and explore the biological functions of the identified lncRNAs. In the screening
phase, we examined the lncRNA expression profile of plasma samples from 24 patients with SLE and
12 healthy controls (HCs) using lncRNA microarray with pooled samples. The candidate lncRNAs
were verified in individual samples by quantitative real-time (qRT)-PCR. In the independent validation
stage, the identified lncRNAs were evaluated in 240 patients with SLE and 120 HCs. The identified
lncRNAs were assessed further in an external validation stage including patients with rheumatoid
arthritis (RA) and primary Sjögren’s syndrome (pSS). In addition, we constructed correlated expression
networks including coding–non-coding co-expression and competing endogenous RNAs (ceRNAs).
Plasma levels of linc0597, lnc0640, and lnc5150 were elevated in SLE patients compared with those of
HCs, whereas levels of GAS5 and lnc7074 were decreased. Five lncRNAs were identified as potential
SLE biomarkers with an area under the receiver operating characteristic curve (AUC) ranging from
0.604 to 0.833 in the independent validation phase. This panel of five lncRNAs had high diagnostic
accuracy for SLE (AUC = 0.966) and distinguished SLE from RA and pSS (AUC = 0.683 and 0.910,
respectively). Co-expression analysis showed that GAS5, lnc0640, and lnc5150 may participate in the
SLE pathogenesis through the MAPK pathway. The ceRNA network indicated that GAS5, lnc0640,
lnc3643, lnc6655, and lnc7074 bind competitively with microRNAs regulating the expression of target
genes. Aberrant expression and related pathways suggest the important role of lncRNAs in SLE
pathogenesis. In addition, the panel of five lncRNAs (GAS5, lnc7074, linc0597, lnc0640, and lnc5150)
in plasma could be used as SLE biomarkers.

Keywords: biomarker; diagnosis; long non-coding RNA; systemic lupus erythematosus

1. Introduction
Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease of variable severity; its
etiology is multifactorial, mainly involving genetic, epigenetic, and environmental factors [1]. It is
a heterogeneous disease and no single clinical characteristic or laboratory test is diagnostic. Lupus
nephritis (LN), as one of the most severe manifestations of SLE, affects up to 70% of SLE patients and
has significant impact on the outcome of SLE patients [2]. The discovery of an effective and reliable
tool for better diagnosis of SLE is essential for disease monitoring and prognostication.

Biomolecules 2019, 9, 206; doi:10.3390/biom9060206 www.mdpi.com/journal/biomolecules

Biomolecules 2019, 9, 206 2 of 16

At present, although the exact pathogenesis of SLE remains unclear, it is known that its etiology
is multifactorial mainly involving genetic, epigenetic, and environmental factors [1,3]. Recently,
several studies have placed a new emphasis on the role of long non-coding RNAs (lncRNAs) with a
length longer than 200 nucleotides and poor protein coding potential [4–6]. It has become apparent
that lncRNAs participate in regulating gene expression by versatile interactions with DNA, RNA,
or proteins [7,8]. It has been reported that lncRNAs play important regulatory roles in Toll-like
receptor signaling, regulating not only the innate immune response but also immune cell development
and adaptive immunity [9,10]. Increasing evidence suggests that lncRNAs play a critical role in the
pathogenesis of autoimmune diseases, such as SLE, rheumatoid arthritis (RA), Sjögren’s syndrome
(SS), etc. [11–15].
The lncRNA growth arrest-specific 5 (GAS5), which is necessary for normal growth arrest,
apoptosis, and cell cycle function in T-cell lines and non-transformed lymphocytes [16], has been
associated with an increased risk for developing SLE in a mouse model [17]. In addition, GAS5 has
been shown to be involved in the development of human SLE [18]. Two other lncRNAs, linc0949 and
linc0597, were decreased in peripheral blood mononuclear cells (PBMCs) from SLE patients. Moreover,
linc0949 could act as a biomarker for SLE [11]. Nuclear-enriched abundant transcript 1 (NEAT1), as an
early lipopolysaccharide response lncRNA, regulates the innate immune response through Toll-like
receptor signaling [19,20]. NEAT1 expression was increased in PBMCs of SLE patients and positively
correlated with disease activity. Moreover, NEAT1 affected the expression of inflammatory chemokines
and cytokines through the activation of the late MAPK signaling pathway [21].
lncRNAs are stable in human plasma and can serve as biomarkers for multiple diseases, such as
cancer, cardiovascular diseases, among others [22–25]. Our previous study showed that a set of plasma
lncRNAs may serve as non-invasive biomarkers for SLE [26]. To understand further the potential of
plasma lncRNAs as biomarkers for SLE, we screened lncRNAs in pooled plasma samples in the present
study from patients with SLE and healthy controls (HCs) using an lncRNA microarray followed by
extensive independent validation.

2. Materials and Methods

2.1. Design and Study Population

The subjects included 264 patients with SLE, 12 HCs, 30 patients with rheumatoid arthritis
(RA), and 31 patients with primary Sjögren’s syndrome (pSS), who were recruited from April 2015
to April 2016. The inclusion and exclusion criteria were as described previously [26]. The disease
severity was quantified according to the Systemic Lupus Erythematosus Disease Activity Index 2000
(SLEDAI-2K) [27]. Disease activity was quantified using the SLEDAI-2K score. Active SLE was defined
as a SLEDAI-2K score >10, whereas those patients with SLEDAI-2K ≤10 were classed as relatively
inactive [11,28].
A four-phase case-control study design was applied. Phase 1 – screening: plasma extracted from
24 new-onset SLE patients (including 12 SLE patients with no LN and 12 LN patients) and from 12 age-
and sex-matched HCs was applied to a human lncRNA microarray. Phase 2 – preliminary validation:
lncRNAs selected from the screening phase were verified in individual samples by a quantitative
real-time (qRT)-PCR assay. Phase 3 – independent validation: the remaining 240 patients with SLE and
120 age- and sex-matched HCs were randomly classified as a training set (160 SLE patients vs. 80 HCs)
and a testing set (80 SLE patients vs. 40 HCs). The HCs were recruited from the physical examination
center of the Second Affiliated Hospital of Anhui Medical University. Phase 4 – external validation: we
examined the levels of candidate lncRNAs considered to be potential candidates for SLE biomarkers
in 30 patients with RA and 31 patients with pSS to identify the specificity. The study procedure was
approved by the Ethics Committee of Anhui Medical University (approval number: 20150115).
Biomolecules 2019, 9, 206 3 of 16

2.2. Total RNA Isolation and qRT-PCR

Total RNA isolation and an lncRNA qRT-PCR assay were conducted as described previously [26].
Total RNA were reverse-transcribed into cDNA with a PrimeScript RT reagent kit (Takara Bio Inc.,
Shiga, Japan), and the quantitative real-time PCR (qPCR) was performed using an SYBR Premix Ex
Taq™ II (Takara Bio Inc.). The relative plasma lncRNA expression level was normalized to the GAPDH
expression [29–31]. The primers used in qRT-PCR of the lncRNAs are listed in Table S1, Supplementary
Materials. The relative expression of lncRNAs was calculated using the 2-∆∆Ct method normalized to
endogenous control, with ∆Ct = Cttarget − Ctreference , − ∆∆Ct = − (sample ∆Ct – control ∆Ct) [32].

2.3. Microarray Analysis of lncRNAs and Data Analysis

In the screening phase, total RNA was isolated from the plasma samples of 12 SLE patients with
LN, 12 SLE patients without LN, and 12 HCs. Four RNA samples were mixed together as a pooled
sample for microarray testing. Accordingly, three pooled RNA samples from each group were used in
microarray screening.
Arraystar Human LncRNA Microarray V3.0 (Arraystar Inc. Rockville, MD, USA) (including
30,586 lncRNAs and 26,109 coding transcripts) was used for the global profiling of human lncRNAs
and protein-coding transcripts. Sample labeling and array hybridization were carried out using the
Agilent One-Color Microarray-Based Gene Expression Analysis (Santa Clara, CA, USA) protocol with
minor modifications. Agilent Feature Extraction software (version 11.0.1.1, Santa Clara, CA, USA) was
applied to analyze the acquired array images. Quantile normalization and subsequent data processing
were conducted using the GeneSpring GX version 12.1 software package (Agilent Technologies, Santa
Clara, CA, USA). The raw microarray data were submitted to the Gene Expression Omnibus public
database (accession number: GSE102547).
The threshold for identifying upregulated and downregulated genes was a p-value ≤0.05 and
a fold change ≥2.0. Hierarchical clustering was conducted to display the distinguishable lncRNA
and mRNA expression patterns among samples. Kyoto Encyclopedia of Genes and Genomes (KEGG)
analysis was performed to describe the roles of the differentially expressed mRNAs.

2.4. Co-Expression Network Analysis

Co-expression analysis was conducted by calculating Pearson’s correlation coefficient (PCC)
between the normalized intensity of the differentially expressed mRNAs and lncRNA biomarkers.
The co-expression network of lncRNAs and mRNAs was drawn using Cytoscape software version
2.8.1 (Cytoscape Consortium, San Diego, CA, USA), with PCC ≥0.935.

2.5. Construction of a Competing Endogenous RNA Network

The construction of a competing endogenous RNA (ceRNA) network included three steps:
(i) screening of microRNAs (miRNAs) relevant to SLE using a combination of miRNAs identified
in the literature and differentially expressed miRNAs found in our previous study; (ii) the putative
interactions of lncRNA–miRNA and miRNA–mRNA were predicted by miRanda version 3.3a [33];
and (iii) on the basis of the predicted miRNA–mRNA and miRNA–lncRNA regulatory pairs, a ceRNA
network was generated in which the lncRNAs and mRNAs interacted via shared miRNAs.

2.6. Statistical Analysis

Normally distributed data are presented as the mean ± standard deviation. Data with a skewed
distribution are expressed as the median (interquartile range). Categorical data are described using
frequency or percentage. A Student’s t-test was used to compare the means of normally distributed
data between groups. A chi-squared test was used to determine the difference in categorical data
between groups. One-way analysis of variance (ANOVA) was applied to quantitative data with
a normal distribution for comparisons among multiple groups (≥3). Logistic regression was used
Biomolecules 2019, 9, 206 4 of 16

to calculate probabilities, odds ratios, and corresponding 95% confidence intervals (CIs). Receiver
operating characteristic (ROC) curves were constructed and the area under the curve (AUC) was
used to evaluate the value of plasma lncRNAs as biomarkers for SLE. A stepwise logistic regression
model was used to select diagnostic lncRNA markers based on the data of the training set or combined
training-and-testing set.
All statistical analyses were conducted using SPSS 10.01 (SPSS Inc., Chicago, IL, USA). Scatter
diagrams were generated through GraphPad Prism 5.01 (GraphPad Software, Inc., San Diego, CA,
USA). ROC curve analysis was conducted by using MedCalc 11.4.2.0 (MedCalc Software, Mariakerke,
Belgium). p-values ≤0.05 were considered statistically significant.

3. Results

3.1. Basic Characteristics of the Study Subjects

In the microarray screening and preliminary validation phases, the demographic and baseline
clinical data of the study subjects were summarized as described previously [26]. The characteristics of
the SLE patients and HCs in the independent validation phase are shown in Table 1. The basic features
of the disease controls (RA and pSS patients) are summarized in Table 2.

Table 1. Basic features of systemic lupus erythematosus (SLE) patients and healthy controls in large-scale
independent validation phase a .

Characteristics Training Set Testing Set

Control subjects 80 40
Age (year) 37 ± 14 34 ± 12
Sex (female/male) 74/6 37/3
Patients with SLE 160 80
Age (year) 38 ± 14 38 ± 12
Sex (female/male) 148/12 74/6
Disease duration (year) 4 (1, 9) 3 (0, 9)
SLEDAI-2K 12 (8, 19) 13 ± 8
Disease manifestations
Renal disease (%) 63 (39) 32 (40)
Vasculitis (%) 6 (4) 4 (5)
Arthritis (%) 64 (40) 26 (33)
Myositis (%) 12 (8) 4 (5)
Rash (%) 77 (48) 35 (44)
Alopecia (%) 67 (42) 26 (33)
Oral ulcer (%) 30 (19) 11 (14)
Pleuritis (%) 18 (11) 8 (10)
Leukopenia (%) 25 (16) 21 (26) *
Thrombocytopenia (%) 43 (27) 19 (24)
Fever (%) 45 (28) 20 (25)
Nervous system disorder (%) 41 (26) 18 (23)
Low complement (%) 108 (68) 52 (65)
Autoantibodies
Anti-dsDNA (%) 71 (44) 27 (34)
Anti-Sm (%) 53 (34) 26 (33)
Anti-SSA (%) 112 (71) 55 (71)
Anti-SSB (%) 26 (17) 14 (18)
Anti-RNP (%) 64 (41) 33 (42)
Anti-Ribosomal P (%) 48 (31) 24 (31)
Medical therapy
Prednisone dose ≥15 mg/day (%) 82 (51) 42 (52)
Prednisone dose <15 mg/day (%) 78 (49) 38 (48)
Antimalarials (%) 127 (79) 57 (71)
Azathioprine, MTX, or CTX (%) 31 (19) 11 (14)
a Normally distributed data were expressed as means ± standard deviation (SD), variables with a skewed
distribution were presented as median (interquartile range). Categorical variable values were described as number (%).
SLEDAI-2K: Systemic Lupus Erythematosus Disease Activity Index 2000; dsDNA: double-stranded DNA; Sm: Smith;
SSA: Sjögren’s syndrome-related antigen A; SSB: Sjögren’s syndrome-related antigen B; RNP: Ribonucleoprotein;
MTX: methotrexate; CTX: cyclophosphamide; * p ≤ 0.05.
Biomolecules 2019, 9, 206 5 of 16

Table 2. Basic features of disease controls (rheumatoid arthritis (RA) patients and primary Sjögren’s
syndrome (pSS) patients) a .

Characteristics RA pSS
Age (year) 53.47 ± 1.63 47.39 ± 9.96
Sex (female/male) 20/10 31/0
Disease duration (year) 7 (3, 19) 5.74 ± 4.40
Medical therapy
Prednisone (%) 20 (67) 27 (87)
Antimalarials (%) 9 (30) 24 (77)
TGP (%) 12 (40) 22 (71)
Leflunomide, MTX, or NSAID (%) 28 (93) 8 (26)
a Normally distributed data were expressed as means ± standard deviation (SD), variables with a skewed distribution

were presented as median (interquartile range). Categorical variable values were described as number (%). TGP:
total glucosides of paeony; MTX: methotrexate; NSAID: nonsteroidal anti-inflammatory drug.

3.2. lncRNA and mRNA Expression Profiles in the Plasma of SLE Patients
Hierarchical clustering showed that there were significant differences in the plasma levels of
lncRNAs and mRNAs between the 24 new-onset SLE patients and the 12 HCs (Figure 1). According
to the criteria of a fold change ≥2 and a p-value ≤0.05, 1315 differentially expressed lncRNAs (743
upregulated and 572 downregulated) and 1363 differentially expressed mRNAs (745 upregulated and
Biomolecules
618 2019, 9, x FORwere
downregulated) PEER REVIEW
identified. 6 of 16

Figure 1. Differential expression of long non-coding RNAs (lncRNAs) and mRNAs between systemic
Figure 1. Differential expression of long non-coding RNAs (lncRNAs) and mRNAs between systemic
lupus
lupuserythematosus
erythematosus (SLE)
(SLE)patients
patients and
andcontrol
controlsubjects.
subjects. Hierarchical
Hierarchical clustering
clustering analysis
analysis of
of (a)
(a)lncRNAs
and (b) mRNAs
lncRNAs and (b) that
mRNAs were differentially
that expressed
were differentially in plasma
expressed in plasmaof SLE
of SLEpatients
patients(SLE1-3: SLE no LN;
(SLE1-3: SLE
LN1-3:
no LN; lupus
LN1-3:nephritis) compared
lupus nephritis) withwith
compared control samples
control samples(C1-3:
(C1-3:control);
control); Hierarchical clustering clearly
Hierarchical clustering
separated SLE (blue
clearly separated SLEbar)
(blueand
bar)normal (red (red
and normal bar)bar)
samples.
samples.Expression
Expressionvalues
values are
are represented
represented inin shades
ofshades of red
red and and green,
green, indicating
indicating expression
expression aboveand
above and below
below thethemedian
median expression
expressionvalue acrossacross all
value
all samples, respectively.
samples, respectively.

3.3. Selection of Candidate lncRNAs and Individual qRT-PCR Confirmation

Candidate lncRNAs identified from the microarray were validated by qRT-PCR according to the
following criteria: (1) when compared with HCs, both SLE patients with and without LN had aberrant
expression of the candidate lncRNAs with a fold change ≥2 and a p-value ≤0.01; (2) the lncRNAs were
acquired from the GENCODE, RefSeq, and UCSC Known Gene databases; (3) average raw signal
intensity >20; and (4) increased/decreased expression of lncRNAs with a fold change ≥10 in both SLE
patients with and without LN.
Ten lncRNAs met the criteria: ENST00000450640 (lnc0640), ENST00000583643 (lnc3643),
Biomolecules 2019, 9, 206 6 of 16

3.3. Selection of Candidate lncRNAs and Individual qRT-PCR Confirmation

Candidate lncRNAs identified from the microarray were validated by qRT-PCR according to the
following criteria: (1) when compared with HCs, both SLE patients with and without LN had aberrant
expression of the candidate lncRNAs with a fold change ≥2 and a p-value ≤0.01; (2) the lncRNAs were
acquired from the GENCODE, RefSeq, and UCSC Known Gene databases; (3) average raw signal
intensity >20; and (4) increased/decreased expression of lncRNAs with a fold change ≥10 in both SLE
patients with and without LN.
Ten lncRNAs met the criteria: ENST00000450640 (lnc0640), ENST00000583643 (lnc3643),
ENST00000584688 (lnc4688), ENST00000425150 (lnc5150), ENST00000506655 (lnc6655), NR_027074
(lnc7074), NST00000507514 (lnc7514), ENST00000458228 (lnc8228), ENST00000519603 (lnc9603), and
uc001agf.1 (lncagf.1) (Table S2, Supplementary Materials). These 10 lncRNAs were verified in individual
samples in the screening phase by qRT-PCR. Of the 10 lncRNAs examined, lnc8228 was not detected
in the plasma of the 24 SLE patients and the 12 HCs, whereas the expression of lnc4688 and lnc9603
did not differ significantly between the two groups. The remaining seven lncRNAs showed aberrant
expression in the plasma of patients with SLE. Compared with HCs, the expression levels of lnc0640,
lnc3643, lnc5150, lnc6655, lnc7514, and lncagf.1 were increased, whereas lnc7074 expression was
decreased in all SLE patients (Table 3).

Table 3. The expressions of 10 candidate long non-coding RNAs (lncRNAs) in the preliminary
validation phase.

Healthy Controls SLE Patients

lncRNAs Z P
(n = 12) (n = 24)
lnc0640 1.00 (0.68, 1.27) 4.52 (3.46, 5.29) −4.832 <0.001
lnc3643 0.97 (0.79, 1.23) 2.69 (1.95, 5.53) −4.799 <0.001
lnc5150 0.74 (0.59, 1.64) 3.01 (1.53, 3.94) −3.893 <0.001
lnc6655 1.05 (0.74, 1.27) 8.35 (5.67, 10.2) −4.832 <0.001
lnc7074 1.00 (0.69, 1.32) 0.24 (0.16, 0.29) −4.832 <0.001
lnc7514 0.71 (0.56, 0.89) 1.69 (1.46, 2.12) −3.356 <0.001
lncagf.1 0.74 (0.44, 1.68) 2.01 (1.21, 2.95) −3.020 0.002
lnc4688 1.05 (0.63, 1.16) 0.93 (0.62, 1.27) −0.201 0.856
lnc9603 0.92 (0.68, 1.34) 0.90 (0.62, 1.06) −0.470 0.655

3.4. Independent Validation of Differentially Expressed Plasma lncRNAs

To verify the expression of these seven lncRNAs and to evaluate their potential value as biomarkers
for SLE and LN, we performed a qRT-PCR assay in an independent validation phase including a
training set and a testing set. A set of plasma lncRNAs (GAS5, linc0597, and lnc-DC) with significantly
different expression between SLE patients and HCs was also included, because our previous study
found that they may serve as novel non-invasive biomarkers for SLE and LN [26].
As shown in Figure 2, in the training set, the expression levels of GAS5, linc0597, lnc-DC, lnc0640,
lnc5150, lnc7074, and lncagf.1 were consistent with the results from the preliminary validation phase
or as described previously [26]. linc0597, lnc0640, and lnc5150 were also increased in all SLE patients
compared with those in the HCs, whereas the levels of GAS5, lnc-DC, and lnc7074 were decreased.
However, unlike the preliminary validation phase, the levels of lnc3643, lnc6655, lnc7514, and lncagf.1
were not significantly different between SLE patients and HCs.
When the 160 SLE patients were divided into 63 with and 97 without LN, the levels of lnc-DC
were significantly higher in LN compared with SLE without nephritis (p = 0.005), but no significant
difference in levels of GAS5 (p = 0.239) and linc0597 (p = 0.252) was found between LN and SLE
without nephritis, which is in agreement with what we observed previously [26]. The levels of lnc-DC,
lnc0640, lnc3643, lnc6655, lnc7074, and lnc7514 were higher in SLE patients with LN compared with
those without LN; however, no significant difference in the levels of GAS5, linc0597, lnc5150, and
lncagf.1 was found between the two groups (Figure S1, Supplementary Materials). Therefore, lncagf.1
was excluded from the testing set.
 As shown in Figure 2, in the training set, the expression levels of GAS5, linc0597, lnc-DC, lnc0640,
lnc5150, lnc7074, and lncagf.1 were consistent with the results from the preliminary validation phase
or as described previously [26]. linc0597, lnc0640, and lnc5150 were also increased in all SLE patients
compared with those in the HCs, whereas the levels of GAS5, lnc-DC, and lnc7074 were decreased.
Biomolecules 2019,
However, 9, 206the preliminary validation phase, the levels of lnc3643, lnc6655, lnc7514,7 and
unlike of 16

lncagf.1 were not significantly different between SLE patients and HCs.

Biomolecules 2019, 9, x FOR PEER REVIEW 8 of 16

with those without LN; however, no significant difference in the levels of GAS5, linc0597, lnc5150,
Figure 2. The expression levels of (a) GAS5, (b) linc0597, (c) lnc-DC, (d) lnc0640, (e) lnc5150, (f) lnc7074,
and lncagf.1
Figure was
2. The found between the two groups (Figure S1,(c)Supplementary Materials). Therefore,
(g) lncagf.1, (h)expression levels
ln3643, (i) lnc7514, of
and (a)(j)GAS5,
lnc6655(b) linc0597,
between systemiclnc-DC, (d) lnc0640,
lupus erythematosus (e)(SLE)
lnc5150, (f)
patients
lncagf.1 was
lnc7074, excluded
(g) from
lncagf.1, (h)the testing
ln3643, (i) set.
lnc7514, and (j) lnc6655 between systemic lupus erythematosus
and healthy controls in the training set.
In(SLE)
the testing
patientsset,
andas shown
healthy in Figure
controls in the 3 and Figure
training set. S2 (Supplementary Materials), the expression
levels of
In the testing set, as shown in Figure 3 and Figure S2lnc6655,
GAS5, linc0597, lnc0640, lnc5150, lnc7074, lnc3643, and lnc7514
(Supplementary were consistent
Materials), with
the expression
the results
levelsWhen from
of GAS5, the
the 160 training
linc0597, set; however,
SLE patients
lnc0640, were
lnc5150, the
divided levels of with
intolnc3643,
lnc7074, 63 lnc-DCanddid97 not
lnc6655, anddiffer
without between
LN,
lnc7514 SLE of
the levels
were patients
lnc-DC
consistent with
and healthy
were controls,
significantly but
higher they
in did
LN differ
compared betweenwith SLE no LN
without patients and
nephritis (pLN
= patients.
0.005),
the results from the training set; however, the levels of lnc-DC did not differ between SLE patients and but no significant
difference in levels
healthy controls, butofthey
GAS5 did (p = 0.239)
differ between and SLElinc0597
no LN(ppatients
= 0.252)andwasLN found between LN and SLE
patients.
without nephritis, which is in agreement with what we observed previously [26]. The levels of lnc-
DC, lnc0640, lnc3643, lnc6655, lnc7074, and lnc7514 were higher in SLE patients with LN compared

Figure 3. The expression levels of (a) linc0597, (b) lnc-DC, (c) GAS5, (d) lnc0640, (e) lnc3643, (f) lnc5150,
(g)
Figurelnc6655,
3. The (h) lnc7074, levels
expression and (i)oflnc7514 between
(a) linc0597, (b)systemic
lnc-DC, lupus erythematosus
(c) GAS5, (d) lnc0640,(SLE) patients (f)
(e) lnc3643, and
healthy controls in the testing set.
lnc5150, (g) lnc6655, (h) lnc7074, and (i) lnc7514 between systemic lupus erythematosus (SLE) patients
and healthy controls in the testing set.
In the combined set, as shown in Figures S3 and S4 (Supplementary Materials), the expression
levels of GAS5,
In the linc0597,
combined lnc-DC,
set, as shownlnc0640, lnc7074,
in Figures lnc3643,
S3 and lnc6655, and lnc7514
S4 (Supplementary werethe
Materials), consistent with
expression
the results
levels from
of GAS5, the training
linc0597, setlnc0640,
lnc-DC, in all comparisons; however,
lnc7074, lnc3643, the levels
lnc6655, of lnc5150
and lnc7514 werewere significantly
consistent with
higher in SLE patients with LN compared with those patients without LN. To further investigate
the results from the training set in all comparisons; however, the levels of lnc5150 were significantly
the stability
higher in SLE of lncRNA
patients expression
with in plasma
LN compared with of SLEpatients
those patients,without
we analyzed
LN. Tothe potential
further influence
investigate theof
stability of lncRNA expression in plasma of SLE patients, we analyzed the potential influence of
disease activity and treatment on their expression level, but found no significant difference (Tables
S3–S7, Supplementary Materials).

3.5. Identification of lncRNAs in Plasma as Potential Biomarkers for SLE

 On the basis of the data from the training set, a discrimination model of an lncRNA panel was
constructed to predict the probability of a diagnosis with SLE by stepwise logistic regression, and
validated in the testing set. The discrimination model of the lncRNA panel was constructed as
follows: logit (P = SLE) = −2.594 + 7.503 × linc0597 − 14.253 × GAS5 + 2.853 × lnc5150 − 7.012 × lnc7074
Biomolecules 2019, 9, 206 8 of 16
+ 6.233 × lnc0640. A patient was classified as “SLE” if logit (P) > 0 according to the logistic regression
model, and as “HC” if logit (P) < 0. The AUC value of the panel of five lncRNAs was 0.966 (95% CI:
disease activity(Figure
0.945–0.987) and treatment
4b). From on their expression
the data level, but
of the testing set,found
the AUCno significant
values of difference
these five (Tables
lncRNAs
S3–S7, Supplementary
(linc0597, GAS5, lnc0640,Materials).
lnc5150, and lnc7074) were 0.649 (95% CI: 0.551-0.746), 0.851 (95% CI: 0.784-
0.917), 0.615 (95% CI: 0.514-0.715), 0.683 (95% CI: 0.587-0.779), and 0.662 (95% CI: 0.563-0.760),
3.5.respectively
Identification of lncRNAs
(Figure 4c). Thein Plasma
AUC ofasthe Potential
panel ofBiomarkers
five lncRNAsfor SLE
was 0.968 (95% CI: 0.939-0.997) (Figure
4d).To evaluate further the potential value as SLE biomarkers of the identified lncRNAs individually
We further
or as a panel, ROC evaluated
curve analysesthe identified lncRNAs
were performed onasdata
biomarkers
from the for SLE individually
training or set,
set, the testing as aandpanel
in a combined set including 240 patients with SLE and 120 HCs. The AUC
the combined set, respectively. From the data of the training set, the AUC values of linc0597, GAS5,values of linc0597, GAS5,
lnc0640,
lnc-DC, lnc5150,
lnc0640, and lnc7074
lnc5150, were 0.640
and lnc7074 were(95%
0.634CI: 0.584-0.696),
(95% 0.833 (95%
CI: 0.566–0.703), CI:(95%
0.824 0.791-0.874), 0.604 (95%
CI: 0.771–0.877),
CI: 0.547-0.661), 0.645 (95% CI: 0.589-0.701), and 0.622 (95% CI: 0.565-0.679), respectively
0.597 (95% CI: 0.527–0.667), 0.598 (95% CI: 0.529–0.668), 0.625 (95% CI: 0.556–0.695), and 0.602 (95% CI: (Figure 4e),
and the AUC value was 0.966
0.531–0.672), respectively (Figure 4a). (95% CI: 0.949-0.984) when the panel of five lncRNAs was evaluated,
which was also significantly higher than the AUC value of GAS5 (Figure 4f).

Figure 4. Performance of individual differentially expressed long non-coding RNAs (lncRNAs) and
Figure 4. Performance of individual differentially expressed long non-coding RNAs (lncRNAs) and
five-lncRNA panel as biomarkers for systemic lupus erythematosus (SLE). (a) The receiver operating
five-lncRNA panel as biomarkers for systemic lupus erythematosus (SLE). (a) The receiver operating
characteristic (ROC) curves of individual differentially expressed lncRNAs in training set; (b) The ROC
characteristic (ROC) curves of individual differentially expressed lncRNAs in training set; (b) The
curve of five-lncRNA panel in training set; (c) The ROC curves of individual differentially expressed
ROC curve of five-lncRNA panel in training set; (c) The ROC curves of individual differentially
lncRNAs in testing set; (d) The ROC curve of five-lncRNA panel in testing set; (e) The ROC curves of
expressed lncRNAs in testing set; (d) The ROC curve of five-lncRNA panel in testing set; (e) The ROC
individual differentially expressed lncRNAs in combined set; (f) The ROC curve of five-lncRNA panel
in combined set.

On the basis of the data from the training set, a discrimination model of an lncRNA panel was
constructed to predict the probability of a diagnosis with SLE by stepwise logistic regression, and
Biomolecules 2019, 9, 206 9 of 16

validated in the testing set. The discrimination model of the lncRNA panel was constructed as follows:
logit (P = SLE) = −2.594 + 7.503 × linc0597 − 14.253 × GAS5 + 2.853 × lnc5150 − 7.012 × lnc7074 + 6.233
× lnc0640. A patient was classified as “SLE” if logit (P) > 0 according to the logistic regression model,
and as “HC” if logit (P) < 0. The AUC value of the panel of five lncRNAs was 0.966 (95% CI: 0.945–0.987)
(Figure 4b). From the data of the testing set, the AUC values of these five lncRNAs (linc0597, GAS5,
lnc0640, lnc5150, and lnc7074) were 0.649 (95% CI: 0.551-0.746), 0.851 (95% CI: 0.784-0.917), 0.615 (95%
CI: 0.514-0.715), 0.683 (95% CI: 0.587-0.779), and 0.662 (95% CI: 0.563-0.760), respectively (Figure 4c).
The AUC of the panel of five lncRNAs was 0.968 (95% CI: 0.939-0.997) (Figure 4d).
We further evaluated the identified lncRNAs as biomarkers for SLE individually or as a panel
in a combined set including 240 patients with SLE and 120 HCs. The AUC values of linc0597, GAS5,
lnc0640, lnc5150, and lnc7074 were 0.640 (95% CI: 0.584-0.696), 0.833 (95% CI: 0.791-0.874), 0.604 (95%
CI: 0.547-0.661), 0.645 (95% CI: 0.589-0.701), and 0.622 (95% CI: 0.565-0.679), respectively (Figure 4e),
and the AUC value was 0.966 (95% CI: 0.949-0.984) when the panel of five lncRNAs was evaluated,
which was also significantly higher than the AUC value of GAS5 (Figure 4f).
Finally, the 240 SLE patients were divided into 95 with and 145 without LN. On the basis of
the data from the independent validation phase, a discrimination model of the lncRNA panel was
also constructed to predict the probability of an LN diagnosis. Stepwise logistic regression and ROC
curve analysis, respectively, were used to explore the potential value of the differentially expressed
lncRNAs as novel biomarkers to distinguish SLE with and without LN both individually and as a
panel. The AUC values are shown in Table 4. The stepwise logistic regression model showed that
lnc-DC, lnc5150, and lnc7514 could be used as a panel of biomarkers to distinguish SLE with LN from
SLE without LN; no other panels with statistical significance were observed. The discrimination model
of the panel of three lncRNAs was constructed to predict the probability of diagnosis with LN as
follows: logit (P = LN) = 0.013 + 1.706 × lnc-DC − 2.053 × lnc5150 + 1.426 × lnc7514, which generated
an AUC of 0.725 (95% CI: 0.659-0.791) (Table 4); however, it was not significantly different compared
with the AUC value of lnc-DC or lnc7514.

Table 4. Performance of individual differentially expressed long non-coding RNAs (lncRNAs) and
three-lncRNA panel as biomarkers for lupus nephritis (LN).

lncRNA AUC Sensitivity Specificity Cutoff Point

lnc-DC 0.683 (0.612–0.753) 0.526 0.807 1.070
lnc0640 0.644 (0.571–0.717) 0.421 0.821 2.250
lnc3643 0.671 (0.600–0.742) 0.526 0.759 1.172
lnc5150 0.601 (0.527–0.675) 0.421 0.772 2.193
lnc6655 0.631 (0.559–0.703) 0.758 0.448 0.607
lnc7074 0.665 (0.594–0.735) 0.632 0.669 0.673
lnc7514 0.684 (0.614–0.755) 0.674 0.662 0.990
three-lncRNA panel 0.725 (0.659–0.791) 0.642 0.766 0.000
AUC: area under curve.

3.6. Plasma Levels of lncRNAs in Patients with SLE, RA, and pSS
In the external validation phase, the levels of these five differentially expressed lncRNAs were
examined in 30 patients with RA and 31 patients with pSS. GAS5 expression was decreased in the
SLE patients from the testing set compared with that in the RA and pSS patients; linc0597 levels were
lower in the SLE patients from the testing set compared with those in the RA patients; lnc7074 levels
were lower in the SLE patients from the testing set compared with those in the pSS patients; and
lnc-DC levels were lower in the SLE patients compared with those in the pSS patients. No significant
difference was found between the SLE and RA patients. Compared with the SLE patients, no significant
differences in the levels of lnc0640 and lnc5150 were found in the RA and pSS patients (Figure 5).
 Biomolecules
Biomolecules 2019,
2019, 9, 9,
206x FOR PEER REVIEW 11ofof1616
10

Figure 5. Validation of differentially expressed long non-coding RNAs (lncRNAs) in systemic lupus
Figure 5. Validation
erythematosus of differentially
(SLE) and expressed
disease controls long non-coding
(rheumatoid RNAs
arthritis (RA) (lncRNAs)
patients in systemic
and primary lupus
Sjögren’s
erythematosus
syndrome (SLE) and(a)
(pSS) patients). disease
GAS5;controls (rheumatoid
(b) linc0597; arthritis
(c) lnc7074; (RA) patients
(d) lnc-DC; and primary
(e) lnc0640; Sjögren’s
(f) lnc5150.
syndrome (pSS) patients). (a) GAS5; (b) linc0597; (c) lnc7074; (d) lnc-DC; (e) lnc0640; (f) lnc5150.
Subsequently, the panel of five lncRNAs from the training set was assessed further in the SLE
patients from the testing
Subsequently, the set andofallfive
panel disease controls,
lncRNAs fromwhich generated
the training set an
wasAUC value further
assessed for the risk score
in the SLE
ofpatients
0.798 (95%
fromCI:the0.723–0.861). Theall
testing set and risk score controls,
disease also significantly discriminated
which generated an AUCthevalue
patients withrisk
for the SLE in
score
the
of testing set from
0.798 (95% the RA and The
CI: 0.723–0.861). the pSSriskpatients,
score alsowith AUC values
significantly of 0.683 (95%
discriminated theCI: 0.570–0.797)
patients andin
with SLE
0.910 (95% CI:
the testing set0.856–0.963),
from the RArespectively
and the pSS(Figure 6).with AUC values of 0.683 (95% CI: 0.570–0.797) and
patients,
0.910 (95% CI: 0.856–0.963), respectively (Figure 6).

3.7. KEGG Pathway Analysis

KEGG pathway analysis identified 11 pathways that were associated with the upregulated
mRNAs and 4 pathways related to the downregulated mRNAs. The “MAPK signaling pathway -
Biomolecules 2019, 9, x FOR PEER REVIEW 12 of 16

Homo sapiens (human)”, “PPAR signaling pathway - Homo sapiens (human)”, and “glycosphingolipid
biosynthesis - lacto and neolacto series - Homo sapiens (human)” were the top three pathways for the
upregulated protein-coding genes, whereas the most enriched network was “axon guidance - Homo
Biomolecules 2019, 9, 206 11 of 16
sapiens (human)” for the downregulated protein-coding genes (Figure S5, Supplementary Materials).

Figure 6.6.Performance
Figure Performanceof five- long non-coding
of five- RNAs (lncRNAs)
long non-coding panel as biomarkers
RNAs (lncRNAs) panel asforbiomarkers
discriminating
for
systemic lupus systemic
discriminating erythematosus
lupus (SLE) from rheumatoid
erythematosus arthritis
(SLE) from (RA) patients
rheumatoid and(RA)
arthritis primary Sjögren’s
patients and
syndrome
primary (pSS) patients.
Sjögren’s syndrome (a) SLE
(pSS)vs. RA; (b)(a)
patients. SLE vs.vs.
SLE pSS;
RA;(c)(b)
SLE vs.vs.
SLE RA + pSS.
pSS; (c) SLE vs. RA + pSS.

3.7. KEGG Pathway Analysis

3.8. Construction of a Co-Expression Network
KEGG pathway analysis identified 11 pathways that were associated with the upregulated mRNAs
Subsequently, in combination with the validation results by qRT-PCR and the data from the
and 4 pathways related to the downregulated mRNAs. The “MAPK signaling pathway—Homo
LncRNA Microarray, we established lncRNA–mRNA co-expression network analysis to explore the
sapiens (human)”, “PPAR signaling pathway—Homo sapiens (human)”, and “glycosphingolipid
biological functions of the differentially expressed lncRNAs in SLE. The lncRNA–mRNA co-
biosynthesis—lacto and neolacto series—Homo sapiens (human)” were the top three pathways for the
expression network analysis revealed that GAS5, lnc0640, lnc5150, lnc7074, lnc3643, lnc6655, and
upregulated protein-coding genes, whereas the most enriched network was “axon guidance—Homo
lnc7514 were correlated highly with 174, 18, 48, 30, 36, 28, and 20 mRNAs, respectively. The network
sapiens (human)” for the downregulated protein-coding genes (Figure S5, Supplementary Materials).
also indicated that the target genes of GAS5, lnc0640, and lnc5150 were dual specificity phosphatase
43.8.
(DUSP4), arrestin
Construction of a beta 2 (ARRB2),
Co-Expression and ribosomal protein S6 kinase A5 (RPS6KA5), respectively,
Network
indicating that the contribution of GAS5, lnc0640, and lnc5150 to the pathogenesis of SLE may be
through Subsequently,
the MAPK in combination
signaling pathway with the validation
(Figures results
S6 and S7, by qRT-PCR
Supplementary and the data from the
Materials).
LncRNA Microarray, we established lncRNA–mRNA co-expression network analysis to explore the
biological
3.9. functions
Construction of aof the differentially
ceRNA Network expressed lncRNAs in SLE. The lncRNA–mRNA co-expression
network analysis revealed that GAS5, lnc0640, lnc5150, lnc7074, lnc3643, lnc6655, and lnc7514 were
According
correlated highlytowith
ceRNA 174,hypothesis, lncRNAs
18, 48, 30, 36, 28, andmay act as a “sponge”
20 mRNAs, of miRNA
respectively. competing
The network with the
also indicated
miRNA response elements (MREs), thereby regulating miRNA-mediated biological
that the target genes of GAS5, lnc0640, and lnc5150 were dual specificity phosphatase 4 (DUSP4),processes [34,35].
To explore
arrestin the
beta underlying
2 (ARRB2), mechanism
and ribosomalofprotein
the differentially
S6 kinase A5expressed lncRNAs
(RPS6KA5), validated
respectively, by qRT-PCR
indicating that
in
the contribution of GAS5, lnc0640, and lnc5150 to the pathogenesis of SLE may be throughnetwork
the pathogenesis of SLE, we used ceRNA analysis in SLE to construct the regulatory the MAPK of
mRNA–miRNA–lncRNA. The ceRNA analysis pointed
signaling pathway (Figures S6 and S7, Supplementary Materials). out that GAS5, lnc0640, lnc3643, lnc6655, and
lnc7074 could act as ceRNAs to regulate the expression of the predicted target genes by competing
for
3.9. common miRNA-binding
Construction sites with mRNAs (Figure S8).
of a ceRNA Network
According to ceRNA hypothesis, lncRNAs may act as a “sponge” of miRNA competing with the
miRNA response elements (MREs), thereby regulating miRNA-mediated biological processes [34,35].
To explore the underlying mechanism of the differentially expressed lncRNAs validated by qRT-PCR
Biomolecules 2019, 9, 206 12 of 16

in the pathogenesis of SLE, we used ceRNA analysis in SLE to construct the regulatory network of
mRNA–miRNA–lncRNA. The ceRNA analysis pointed out that GAS5, lnc0640, lnc3643, lnc6655, and
lnc7074 could act as ceRNAs to regulate the expression of the predicted target genes by competing for
common miRNA-binding sites with mRNAs (Figure S8).

4. Discussion
In the present study, we investigated the expression profile of plasma lncRNAs in SLE patients,
and found that five lncRNAs (linc0597, GAS5, lnc0640, lnc5150, and lnc7074) were aberrantly expressed
as compared with HCs. Then, we evaluated the value of these five lncRNAs as potential biomarkers
for SLE. Finally, we explored the potential targets and mechanism of aberrantly expressed lncRNAs
using bioinformatics.
During the preliminary screening stage of the present study, 10 differentially expressed candidate
lncRNAs were selected for preliminary validation and independent validation. Compared with HCs,
the plasma levels of lnc0640 and lnc5150 were significantly elevated, whereas lnc7074 expression was
significantly decreased. In accordance with our previous studies, linc0597 and GAS5 were differentially
expressed in both the training and testing sets; however, there was no significant change in lnc-DC
expression in the testing set, which may be due to the small sample size.
On the basis of the results of independent validation with a large sample size, we investigated
the plasma levels of lncRNAs in SLE patients with and without LN in both training and testing
sets. The expression levels of lnc-DC, lnc0640, lnc3643, lnc5150, lnc6655, lnc7074, and lnc7514 were
significantly higher in the patients with LN compared with those without LN, suggesting that the
plasma levels of these seven lncRNAs may reflect kidney pathology in SLE.
Our recent study suggested that plasma levels of linc0597 and GAS5 may serve as potential
biomarkers for SLE; lnc-DC could serve as a biomarker for distinguishing SLE with LN from SLE
without LN [26]. Available evidence has indicated that circulating lncRNAs are stable in the plasma,
because they are resistant to digestion from endogenous RNases [31,36–38]. In addition, a large number
of studies have demonstrated that lncRNAs may serve as biomarkers for the diagnosis of cancer,
cardiovascular diseases, among others. [22–25]. Therefore, we evaluated the value of these differentially
expressed lncRNAs as biomarkers for SLE. Our results showed that GAS5, lnc7074, linc0597, lnc0640,
and lnc5150 could be potential biomarkers for SLE. According to the expression of lncRNAs in RA and
pSS patients, GAS5, lnc7074, and linc0597 demonstrated better specificity than the other two lncRNAs.
The combination of these five lncRNAs in a logistic regression model demonstrated a higher AUC
value than when they were used individually. Additionally, the combination of these five lncRNAs
could distinguish SLE from RA and pSS. We investigated the value of lnc-DC, lnc0640, lnc3643, lnc5150,
lnc6655, lnc7074, and lnc7514 as biomarkers for LN individually and as a panel, and found that the
panel of lnc-DC, lnc5150, and lnc7514 did not significantly increase the AUC value compared with
when they were used individually.
Currently, the function of most lncRNAs and their mechanism in disease pathogenesis are not clear.
Therefore, we explored the potential targets and mechanism of the differentially expressed lncRNAs by
bioinformatics. KEGG analysis demonstrated the association of the MAPK signaling pathway with
SLE. It has been confirmed that the MAPK signaling pathway can regulate the immune response of T
cells and B cells as well as the production of multiple SLE-related inflammatory factors, such as TNF-α,
IL-1/6, and IFN [39,40]. Molad et al. reported that the activity of two members of the MAPK family,
namely, ERK and JNK, are associated with SLE disease activity [40]. lncRNA–mRNA co-expression
network analysis showed that GAS5, lnc0640, and lnc5150 may participate in the development of SLE
through the MAPK signaling pathway; DUSP4 (also called MKP2) may be a positively regulated target
gene of GAS5, ARRB2 may be a positively regulated target gene of lnc0640, and RPS6KA5 (also called
MSK1) may be a positively regulated target gene of lnc5150. Furthermore, we predicted the target
genes of the lncRNAs and their regulatory mechanism, and the results showed that GAS5, lnc0640,
lnc3643, lnc6655, and lnc7074 could act as ceRNAs to regulate the expression of the predicted target
Biomolecules 2019, 9, 206 13 of 16

genes by acting as “sponges” for the target miRNAs. Increasing evidence has shown that lncRNAs can
function as ceRNAs in distinct physiological and pathophysiological states. Many validated ceRNA
pairs participate in the initiation and progression of cancers, and systemic ceRNA network analyses
revealing the potential of ceRNAs in diagnosis, therapy, and prognosis of cancers have also been
performed [41]. Currently, the exact role of the ceRNA network in SLE remains undefined, and further
studies are awaited to experimentally validate the true ceRNAs for certain non-coding transcripts and
provide functional information for these non-coding RNAs. Dissecting the ceRNA network in SLE is
likely to enhance our understanding of the roles of transcriptome, particularly non-coding transcripts,
and enrich our knowledge of the pathogenesis, diagnosis, and therapy of this disease.
Nevertheless, several limitations should be noted in the present study. First, all the study subjects
were recruited from two tertiary hospitals, and the sample size was somewhat small, which may restrict
the generalizability of our results. Second, it would be beneficial to know the prediction accuracy
that would be obtained when comparing the model with clinical data and with clinical data plus a
biomarker panel. However, since most clinical parameters (rheumatoid factor, anti-CCP antibodies,
anti-Ro/La, anti-dsDNA, etc.) were not available for healthy controls, we could thus not compare the
model with clinical data and with clinical data plus a biomarker panel. Therefore, further studies with
a large sample size in different populations are needed to confirm our findings.

5. Conclusions
Taken together, the plasma levels of the panel of five lncRNAs (GAS5, lnc7074, linc0597, lnc0640,
and lnc5150) may serve as biomarkers for SLE. GAS5, lnc0640, and lnc5150 may participate in the
development of SLE through the MAPK signaling pathway. In addition, GAS5, lnc0640, lnc3643,
lnc6655, and lnc7074 could act as ceRNAs to regulate the expression of the predicted target genes by
acting as “sponges” for the target miRNAs. Future studies are needed to further unveil the exact role
of these lncRNAs and their ceRNA network in SLE.

Supplementary Materials: The following are available online at http://www.mdpi.com/2218-273X/9/6/206/s1.

Figure S1: The expression levels of GAS5, linc0597, lnc-DC, lnc0640, lnc5150, lnc7074, lncagf.1, ln3643, lnc7514,
and lnc6655 between SLE no LN patients and LN patients in the training set; Figure S2: The expression levels of
linc0597, lnc-DC, GAS5, lnc0640, lnc3643, lnc5150, lnc6655, lnc7074, and lnc7514 between SLE no LN patients
and LN patients in the testing set; Figure S3: The expression levels of linc0597, lnc-DC, GAS5, lnc0640, lnc3643,
lnc5150, lnc6655, lnc7074, and lnc7514 between SLE patients and healthy controls in the combined set; Figure
S4: The expression levels of linc0597, lnc-DC, GAS5, lnc0640, lnc3643, lnc5150, lnc6655, lnc7074, and lnc7514
between SLE no LN patients and LN patients in the combined set; Figure S5: Differentially expressed mRNAs
with significant enrichment score of pathway: (a) upregulated mRNAs; (b) downregulated mRNAs; Figure S6:
Co-expression network of associated mRNAs with GAS5 (ENST00000449289); Figure S7: Co-expression network of
associated mRNAs with individual lncRNAs of lnc0640 (ENST00000450640), lnc3643 (ENST00000583643), lnc5150
(ENST00000425150), lnc6655 (ENST00000506655), lnc7074 (NR_027074), and lnc7514 (ENST00000507514); Figure
S8: ceRNA networks of GAS5(ENST00000449289), lnc0640 (ENST00000450640), lnc3643 (ENST00000583643),
lnc6655 (ENST00000506655), and lnc7074 (NR_027074); Table S1: Primer sequences used for qPCR; Table S2:
Ten differential expression plasma candidate lncRNAs with SLE by microarray; Table S3: Associations of the
expression of plasma lnc7074 with categorical clinical parameters of SLE patients; Table S4: Associations of the
expression of plasma linc0597 with categorical clinical parameters of SLE patients; Table S5: Associations of the
expression of plasma lnc0640 with categorical clinical parameters of SLE patients; Table S6: Associations of the
expression of plasma lnc05150 with categorical clinical parameters of SLE patients; Table S7: Associations of the
expression of plasma GAS5 with categorical clinical parameters of SLE patients.
Author Contributions: Conceptualization, D.-Q.Y. and H.-F.P.; methodology, H.-F.P. and G.-C.W.; software,
G.-C.W. and Y.H.; validation, H.-F.P. and D.-Q.Y.; formal analysis, G.-C.W.; investigation, G.-C.W., Y.H., and S.-Y.G.;
resources, D.-Q.Y. and H.-F.P.; data curation, D.-Q.Y. and H.-F.P.; writing—original draft preparation, G.-C.W. and
Y.H.; writing—review and editing, H.-F.P. and G.-C.W.; supervision, D.-Q.Y. and H.-F.P.; project administration,
D.-Q.Y. and H.-F.P.; funding acquisition, D.-Q.Y. and H.-F.P.
Funding: This work was funded by the National Natural Science Foundation of China (81573222, 81872687) and
the Anhui Provincial Natural Science Foundation (1908085MH294).
Conflicts of Interest: The authors declare no conflict of interest.
Biomolecules 2019, 9, 206 14 of 16

References
1. Rahman, A.; Isenberg, D.A. Systemic lupus erythematosus. N. Engl. J. Med. 2008, 358, 929–939. [CrossRef]
[PubMed]
2. Borchers, A.T.; Leibushor, N.; Naguwa, S.M.; Cheema, G.S.; Shoenfeld, Y.; Gershwin, M.E. Lupus nephritis:
A critical review. Autoimmun. Rev. 2012, 12, 174–194. [CrossRef] [PubMed]
3. Teruel, M.; Alarcon-Riquelme, M.E. The genetic basis of systemic lupus erythematosus: What are the risk
factors and what have we learned. J. Autoimmun. 2016, 74, 161–175. [CrossRef]
4. Perkel, J.M. Visiting “noncodarnia”. Biotechniques 2013, 54, 303–304. [CrossRef] [PubMed]
5. Wang, P.; Xue, Y.; Han, Y.; Lin, L.; Wu, C.; Xu, S.; Jiang, Z.; Xu, J.; Liu, Q.; Cao, X. The STAT3-binding long
noncoding RNA lnc-DC controls human dendritic cell differentiation. Science 2014, 344, 310–313. [CrossRef]
6. Atianand, M.K.; Hu, W.; Satpathy, A.T.; Shen, Y.; Ricci, E.P.; Alvarez-Dominguez, J.R.; Bhatta, A.;
Schattgen, S.A.; McGowan, J.D.; Blin, J.; et al. A long noncoding RNA lincRNA-EPS acts as a transcriptional
brake to restrain inflammation. Cell 2016, 165, 1672–1685. [CrossRef] [PubMed]
7. Zhang, Y.; Cao, X. Long noncoding RNAs in innate immunity. Cell. Mol. Immunol. 2016, 13, 138–147.
[CrossRef]
8. Bonasio, R.; Shiekhattar, R. Regulation of transcription by long noncoding RNAs. Annu. Rev. Genet. 2014, 48,
433–455. [CrossRef] [PubMed]
9. Wu, G.C.; Pan, H.F.; Leng, R.X.; Wang, D.G.; Li, X.P.; Li, X.M.; Ye, D.Q. Emerging role of long noncoding
RNAs in autoimmune diseases. Autoimmun. Rev. 2015, 14, 798–805. [CrossRef]
10. Murphy, M.B.; Medvedev, A.E. Long noncoding RNAs as regulators of Toll-like receptor signaling and innate
immunity. J. Leukoc. Biol. 2016, 99, 839–850. [CrossRef] [PubMed]
11. Wu, Y.; Zhang, F.; Ma, J.; Zhang, X.; Wu, L.; Qu, B.; Xia, S.; Chen, S.; Tang, Y.; Shen, N. Association of
large intergenic noncoding RNA expression with disease activity and organ damage in systemic lupus
erythematosus. Arthritis Res. Ther. 2015, 17, 131. [CrossRef] [PubMed]
12. Muller, N.; Doring, F.; Klapper, M.; Neumann, K.; Schulte, D.M.; Turk, K.; Schroder, J.O.; Zeuner, R.A.;
Freitag-Wolf, S.; Schreiber, S.; et al. Interleukin-6 and tumour necrosis factor-alpha differentially regulate
lincRNA transcripts in cells of the innate immune system in vivo in human subjects with rheumatoid arthritis.
Cytokine 2014, 68, 65–68. [CrossRef] [PubMed]
13. Lu, M.C.; Yu, H.C.; Yu, C.L.; Huang, H.B.; Koo, M.; Tung, C.H.; Lai, N.S. Increased expression of long
noncoding RNAs LOC100652951 and LOC100506036 in T cells from patients with rheumatoid arthritis
facilitates the inflammatory responses. Immunol. Res. 2016, 64, 576–583. [CrossRef] [PubMed]
14. Sandhya, P.; Joshi, K.; Scaria, V. Long noncoding RNAs could be potential key players in the pathophysiology
of Sjogren’s syndrome. Int. J. Rheum. Dis. 2015, 18, 898–905. [CrossRef] [PubMed]
15. Wang, J.; Peng, H.; Tian, J.; Ma, J.; Tang, X.; Rui, K.; Tian, X.; Wang, Y.; Chen, J.; Lu, L.; et al. Upregulation of
long noncoding RNA TMEVPG1 enhances T helper type 1 cell response in patients with Sjogren syndrome.
Immunol. Res. 2016, 64, 489–496. [CrossRef] [PubMed]
16. Mourtada-Maarabouni, M.; Hedge, V.L.; Kirkham, L.; Farzaneh, F.; Williams, G.T. Growth arrest in human
T-cells is controlled by the non-coding RNA growth-arrest-specific transcript 5 (GAS5). J. Cell Sci. 2008, 121,
939–946. [CrossRef] [PubMed]
17. Haywood, M.E.; Rose, S.J.; Horswell, S.; Lees, M.J.; Fu, G.; Walport, M.J.; Morley, B.J. Overlapping BXSB
congenic intervals, in combination with microarray gene expression, reveal novel lupus candidate genes.
Genes Immun. 2006, 7, 250–263. [CrossRef]
18. Suarez-Gestal, M.; Calaza, M.; Endreffy, E.; Pullmann, R.; Ordi-Ros, J.; Sebastiani, G.D.; Ruzickova, S.;
Jose Santos, M.; Papasteriades, C.; Marchini, M.; et al. Replication of recently identified systemic lupus
erythematosus genetic associations: A case-control study. Arthritis Res. Ther. 2009, 11, R69. [CrossRef]
19. Imamura, K.; Imamachi, N.; Akizuki, G.; Kumakura, M.; Kawaguchi, A.; Nagata, K.; Kato, A.; Kawaguchi, Y.;
Sato, H.; Yoneda, M.; et al. Long noncoding RNA NEAT1-dependent SFPQ relocation from promoter region
to paraspeckle mediates IL8 expression upon immune stimuli. Mol. Cell 2014, 53, 393–406. [CrossRef]
20. Naganuma, T.; Nakagawa, S.; Tanigawa, A.; Sasaki, Y.F.; Goshima, N.; Hirose, T. Alternative 30 -end
processing of long noncoding RNA initiates construction of nuclear paraspeckles. EMBO J. 2012, 31,
4020–4034. [CrossRef]
Biomolecules 2019, 9, 206 15 of 16

21. Zhang, F.; Wu, L.; Qian, J.; Qu, B.; Xia, S.; La, T.; Wu, Y.; Ma, J.; Zeng, J.; Guo, Q.; et al. Identification of the
long noncoding RNA NEAT1 as a novel inflammatory regulator acting through MAPK pathway in human
lupus. J. Autoimmun. 2016, 75, 96–104. [CrossRef] [PubMed]
22. Yang, Y.; Cai, Y.; Wu, G.; Chen, X.; Liu, Y.; Wang, X.; Yu, J.; Li, C.; Jose, P.A.; Zhou, L.; et al. Plasma long
non-coding RNA, CoroMarker, a novel biomarker for diagnosis of coronary artery disease. Clin. Sci. (Lond.)
2015, 129, 675–685. [CrossRef] [PubMed]
23. Kumarswamy, R.; Bauters, C.; Volkmann, I.; Maury, F.; Fetisch, J.; Holzmann, A.; Lemesle, G.; de Groote, P.;
Pinet, F.; Thum, T. Circulating long noncoding RNA, LIPCAR, predicts survival in patients with heart failure.
Circ. Res. 2014, 114, 1569–1575. [CrossRef] [PubMed]
24. Tang, J.; Jiang, R.; Deng, L.; Zhang, X.; Wang, K.; Sun, B. Circulation long non-coding RNAs act as biomarkers
for predicting tumorigenesis and metastasis in hepatocellular carcinoma. Oncotarget 2015, 6, 4505–4515.
[CrossRef]
25. Ren, S.; Wang, F.; Shen, J.; Sun, Y.; Xu, W.; Lu, J.; Wei, M.; Xu, C.; Wu, C.; Zhang, Z.; et al. Long non-coding
RNA metastasis associated in lung adenocarcinoma transcript 1 derived miniRNA as a novel plasma-based
biomarker for diagnosing prostate cancer. Eur. J. Cancer 2013, 49, 2949–2959. [CrossRef] [PubMed]
26. Wu, G.C.; Li, J.; Leng, R.X.; Li, X.P.; Li, X.M.; Wang, D.G.; Pan, H.F.; Ye, D.Q. Identification of long non-coding
RNAs GAS5, linc0597 and lnc-DC in plasma as novel biomarkers for systemic lupus erythematosus.
Oncotarget 2017, 8, 23650–23663. [CrossRef] [PubMed]
27. Gladman, D.D.; Ibanez, D.; Urowitz, M.B. Systemic lupus erythematosus disease activity index 2000.
J. Rheumatol. 2002, 29, 288–291.
28. Hayakawa, I.; Hasegawa, M.; Matsushita, T.; Yanaba, K.; Kodera, M.; Komura, K.; Takehara, K.; Sato, S.
Increased cutaneous T-cell-attracting chemokine levels in sera from patients with systemic sclerosis.
Rheumatology (Oxford) 2005, 44, 873–878. [CrossRef] [PubMed]
29. Wang, Y.; Li, Z.; Zheng, S.; Zhou, Y.; Zhao, L.; Ye, H.; Zhao, X.; Gao, W.; Fu, Z.; Zhou, Q.; et al. Expression
profile of long non-coding RNAs in pancreatic cancer and their clinical significance as biomarkers. Oncotarget
2015, 6, 35684–35698. [CrossRef]
30. Wang, Y.; Zhao, X.; Ju, W.; Flory, M.; Zhong, J.; Jiang, S.; Wang, P.; Dong, X.; Tao, X.; Chen, Q.; et al.
Genome-wide differential expression of synaptic long noncoding RNAs in autism spectrum disorder.
Transl. Psychiatry 2015, 5, e660. [CrossRef]
31. Tong, Y.S.; Wang, X.W.; Zhou, X.L.; Liu, Z.H.; Yang, T.X.; Shi, W.H.; Xie, H.W.; Lv, J.; Wu, Q.Q.; Cao, X.F.
Identification of the long non-coding RNA POU3F3 in plasma as a novel biomarker for diagnosis of
esophageal squamous cell carcinoma. Mol. Cancer 2015, 14, 3. [CrossRef] [PubMed]
32. Schmittgen, T.D.; Livak, K.J. Analyzing real-time PCR data by the comparative C(T) method. Nat. Protoc.
2008, 3, 1101–1108. [CrossRef] [PubMed]
33. John, B.; Enright, A.J.; Aravin, A.; Tuschl, T.; Sander, C.; Marks, D.S. Human Micro RNA targets. PLoS Biol.
2004, 2, e363. [CrossRef]
34. Mercer, T.R.; Dinger, M.E.; Mattick, J.S. Long non-coding RNAs: Insights into functions. Nat. Rev. Genet.
2009, 10, 155–159. [CrossRef] [PubMed]
35. Salmena, L.; Poliseno, L.; Tay, Y.; Kats, L.; Pandolfi, P.P. A ceRNA hypothesis: the Rosetta Stone of a hidden
RNA language? Cell 2011, 146, 353–358. [CrossRef]
36. Mohankumar, S.; Patel, T. Extracellular vesicle long noncoding RNA as potential biomarkers of liver cancer.
Brief Funct. Genomics 2016, 15, 249–256. [CrossRef]
37. Keller, S.; Sanderson, M.P.; Stoeck, A.; Altevogt, P. Exosomes: from biogenesis and secretion to biological
function. Immunol. Lett. 2006, 107, 102–108. [CrossRef]
38. Dong, L.; Lin, W.; Qi, P.; Xu, M.D.; Wu, X.; Ni, S.; Huang, D.; Weng, W.W.; Tan, C.; Sheng, W.; et al. Circulating
long RNAs in serum extracellular vesicles: their characterization and potential application as biomarkers for
diagnosis of colorectal cancer. Cancer Epidemiol. Biomarkers Prev. 2016, 25, 1158–1166. [CrossRef]
39. Yang, J.; Lu, Y.W.; Lu, M.M.; Leng, R.X.; Pan, H.F.; Ye, D.Q. MicroRNA-101, mitogen-activated protein kinases
and mitogen-activated protein kinases phosphatase-1 in systemic lupus erythematosus. Lupus 2013, 22,
115–120. [CrossRef] [PubMed]
Biomolecules 2019, 9, 206 16 of 16

40. Molad, Y.; Amit-Vasina, M.; Bloch, O.; Yona, E.; Rapoport, M.J. Increased ERK and JNK activities correlate
with disease activity in patients with systemic lupus erythematosus. Ann. Rheum. Dis. 2010, 69, 175–180.
[CrossRef] [PubMed]
41. Li, L.J.; Zhao, W.; Tao, S.S.; Leng, R.X.; Fan, Y.G.; Pan, H.F.; Ye, D.Q. Competitive endogenous RNA network:
Potential implication for systemic lupus erythematosus. Expert Opin. Ther. Targets 2017, 21, 639–648.
[CrossRef] [PubMed]

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